Soybean (Glycine max) provides an excellent source of dietary protein and fiber and is a good source of calcium, iron, zinc, phosphate, magnesium, B vitamins, and folate. It contains all the essential amino acids required in the human diet and, if ingested in the proper amount, soybean can replace animal protein as the sole source of dietary protein (Young, Journal of the American Dietetic Association, 91:828-36, 1991). Because of its high nutritional value, the use of soy protein in food products has increased. In addition to being eaten whole, soybean seed is a versatile food source that can be processed into multiple products including tofu, cheese, meat alternatives, textured soy protein, tempeh, and yogurt. Over the past 10 years, soy's versatility has led to the entry of more than 2,000 new soy food products into the market place (Marsh Laux, Soyfoods Industry Profile, Agricultural Marketing Resource Center, Iowa State University, January 2003). Soy is also used to make infant formula, which is now widely used in the United States. Of those infants in the United States who are fed formula, approximately twenty-five percent or 750,000 consume soy formula. It is particularly valuable for children who are lactose-intolerant or allergic to cow's milk.
Soy-protein foods, including infant formula, are high in the isoflavones genistein and daidzein and their glycosides. These phytoestrogens are nonsteroidal compounds that are similar in both structure and action to estrogen and have been associated with the prevention of menopausal symptoms and hormone-related chronic diseases, including cancer and cardiovascular disease. Though the consumption of phytoestrogens may have beneficial effects in adults, these bioactive compounds exert a wide range of both hormone-dependent and hormone-independent physiological actions and their health effects in infants, who normally have low circulating hormone levels, are not well understood.
In infants who are fed exclusively soy formula, plasma isoflavone concentrations are 13,000- to 22,000-fold higher than plasma estradiol levels early in life and the daily intake of isoflavones in soy-fed infants is five- to ten-fold higher (relative to body weight) than the level shown to affect the menstrual cycle of adult women (Setchell et al., Am J. Clin. Nutr. 68(suppl):1453S-1461S, 1998). To put these values into context, it has been suggested that children fed solely soy milk are consuming the hormonal equivalent of five birth control pills per day. Because exposure to pharmacologically active compounds such as isoflavones could have adverse developmental effects, the ingestion of soy formula by infants raises important health concerns.
Studies in rodent models have shown that significant changes to the thymus and immune system occur in mice following genistein treatment that results in plasma levels comparable to those reported in soy-fed infants (Yellayi et al., Proc. Natl. Acad. Sci. U.S.A 99:7616-7621, 2002). Consistent with these findings, both gamma globulins and immunoglobulins are decreased in soy-fed infants compared to infants who are fed cow's milk and, as a result, soy-fed infants show a higher prevalence of asthma and allergies. Additionally, women who were fed soy products as infants show a 90% increase in the regular use of allergy medicine suggesting that there may be long-term consequences to soy ingestion early in life. In addition to affecting immune function, isoflavones, in particular genistein, inhibit thyroid peroxidase, the enzyme which makes the hormones T3 and T4, and isoflavones can disrupt thyroid function, causing hypothyroidism and goiter. The observation that infants who ingest soy products are at increased risk for developing autoimmune thyroid disease suggests that exposure to high levels of isoflavones in early life may affect the development of autoimmune diseases.
Estrogen exposure during critical stages of development disrupts reproductive and endocrine systems raising concerns that estrogenic actions of soy isoflavones may have similar long-term adverse effects in infants. During development, extreme changes in estrogen levels cause long-term changes in the reproductive organs of both males and females. Upon sexual maturation, men exhibit smaller testes than normal. In females, premature maturation is a consequence causing both physiological and psychological difficulties. Further, later in life these women may experience reproductive problems such as failure to menstruate and infertility as well as increased risk of breast cancer. Findings like these have led some physicians and scientists to suggest that soy products be used with caution, particularly during infancy and early childhood. In 2003, the Paediatric Group of the British Dietetic Association recommended that dieticians discourage the use of soy protein during the first six months of life when phytoestrogens are most likely to exert developmental effects (http://www.bda.uk.com/). The Paediatric policy of the Royal Australian College of Physicians cautions against the unnecessary use of soy formula stating that there is some evidence that soy formula may impair immunity and emphasizing that the long-term effects of soy contaminants on health are unknown (http://www.racp.edu.au/hpu/paed/soy/index.htm).
Soy food products have many nutritional advantages and soy-based baby food and formula with reduced isoflavone content would limit the exposure of children to the possible harmful effects associated with phytoestrogens. This may be achieved by reducing the enzyme isoflavone synthase in the soybeans used to make formula, thereby limiting their synthesis of isoflavones. This enzyme catalyzes the conversion of liquiritigenin and naringenin to the phytoestrogens daidzein and genistein, respectively (Humphreys et al., Trends in Plant Science, 5(7); 271-272, 2000). Two genes encoding isoflavone synthases (IFS), isoflavone synthase 1 (IFS1) and isoflavone synthase 2 (IFS2), have been identified in soybean to date (Jung et al., Nature Biotechnology 18(2):208-212, 2000) and it is possible that additional genes for this enzyme exist in soybean. The reduction of isoflavones from soy would decrease some of the adverse effects that are thought to occur when isoflavone-containing soy products are ingested, especially early in life. Soy products made from soybeans with reduced phytoestrogens would provide consumers with food alternatives that may better suit their personal health needs.
Soybean lines with varying isoflavone levels have been developed using standard breeding methods. Some lines have lower levels, the isoflavone levels in the reduced lines still represent a significant amount and it would be useful to have soybean cultivars with even greater reductions in their isoflavone content.
Genetic engineering can be used to modify expression of particular genes and such techniques have been utilized successfully to increase isoflavone production in soybean (Yu et al., Plant Physiol. 124:781-793, 2000; Jung et al., Nature Biotechnology 18:208-212, 2000; WO 00/44909; U.S. Pat. No. 6,521,433; WO 03/072790 A2; US 20030150012 A1; US 20040128711; US 20040006795). However, there have been no reports to date that isoflavone content of soybean seed can be significantly reduced utilizing any method, including standard breeding, mutagenesis or transgenic technology. Subramanian et al. (Plant Physiol. 137:1345-1353, 2005) reported that isoflavone accumulation in cotyledon tissues in response to a pathogen was reduced following their transformation with an RNAi construct to silence ISF1 and ISF2. However, the transformed tissues were not grown into plants and the ability of this genetic engineering to reduce isoflavone content of soybean seeds has never been evaluated. In addition, public acceptance of genetically modified plants, particularly with respect to plants used for food, is low. Therefore, it would be useful to have novel soybean cultivars that have further reduced levels of phytoestrogens in their seeds and in particular, those having reduced levels of phytoestrogens in their seeds due to alterations in isoflavone synthases that are not the result of genetic engineering.